Ms of size and granularity. By day 12, about 90 of cells were positive for both p63 and CK14, but the percentage of CK5 was lowest among the three 3-Bromo-5-chloro-2-fluoroaniline antigens. Of the cells that survived on day 4, 69 ?8 were p63+, 4 ?2.5 were CK5+, and 4.9 ?3 were PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/14960617 CK14+. This finding suggests that all p63+ cells may have survived, and some of them seemed to have differentiated into keratinocyte lineage with CK5 and CK14 expression by day 4. Obviously, specific KPC adhesion to the FC occurred, and floating cells were eliminated from the culture over a period of 4 days. The higher percentage of p63+ cells on day 4 as compared with day 1 may be due to the elimination of largenumber of nonspecific cells from the culture and proliferation of p63+ KPCs. A remarkable difference was seen in CK5 and CK14 expression on cells cultured for 8 days. Whereas p63+ cells were about 80 of the total viable population, 2,2,3,3-Tetrafluoropropyl N,N'-diethylcarbamimidothioate trifluoromethanesulfonate >95 were CK5+, and >80 were CK14+. Average ?standard deviation (SD) of the values from three donors (Figure 2A) show similar progression of expression for p63, CK5, and CK14, with the lowest SD by day 12 of culture. A differentiation experiment was done by using PBMNCs from many other donors for confirming differentiation; however, tracking and quantification of marker expression by using three donors only is presented here. Overall, it was observed that if the PBMNC yield was good, the cell survival and expression of markers was also as good, as the data show in Figure 2. But if PBMNC yield and seeding density were poor, cell survival and marker expression were poor, too. It appeared that seeding density is crucial for KPC survival, with the best results when the seeding density of leukocytes was about 1 ?106/cm2 of the biomimetic matrix. In Figure 2B, results of the gene-expression analysis are shown. In ijms17122034 cells on day 1, one isoform of p63 (TAp63) wasNair and Krishnan Stem Cell Research Therapy 2013, 4:38 http://stemcellres.com/content/4/2/Page 6 ofFigure 2 Evidence for differentiation. (A) Compiled data of flow-cytometric analysis during cell culture. The percentage of cells positive for p63, CK5, and CK14 on each day of analysis during the periods of culture is presented. Periods of analysis against the percentage of positive cells for each parameter are marked in the respective axis. Average ?SD of replicate PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/9282946 experiments (n = 3), carried out by using different donor PBMNCs are presented. The values are plotted in the bar diagram for comparison of percentage of positive cells on each day. CK14- and CK5-positive cells were absent on day 1. (B) Expressions of p63, CK5, and CK14 genes on the day of starting the culture and after 12 days of culture. GAPDH was the housekeeping gene, and TAp63 was the only KPC-specific gene that amplified on day 1. Np63, CK5, and CK14 were expressed on day 12, and the products showed specific molecular size.expressed, but none of the other tested markers was amplified in 40 cycles of reaction. In cells on day 12, TAp63, Np63, CK5, and CK14 genes were clearly expressed. These data confirm that p63, CK5, and CK14 are expressed when PBMNCs were cultured on the KPC-specific niche in this study. How cells were stimulated to the keratinocyte lineage during the later period of cell culture is beyond the scope of this study. It is likely that the molecules present on the culture matrix and in the medium together would have influenced the keratinocyte lineage commitment and expression of more keratinocyte markers. The KPCs were.